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Endocrinology Vol. 144, No. 11 5050-5057
Copyright © 2003 by The Endocrine Society

The Positive Inotropic and Calcium-Mobilizing Effects of Growth Hormone-Releasing Peptides on Rat Heart

Xiang-Bin Xu, Ji-Min Cao, Jing-Jiang Pang, Rong-Kun Xu, Chao Ni, Wen-Ling Zhu, Kamlesh Asotra, Meng-Chin Chen and Chen Chen

Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College (X.-B.X., J.-M.C., J.-J.P., R.-K.X., M.-C.C.), Beijing 100005, China; Division of Cardiology, Department of Medicine, Peking Union Hospital (C.N., W.-L.Z.), Beijing 100005, China; Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center (K.A.), Los Angeles, California 90048; and Endocrine Cell Biology, Prince Henry’s Institute of Medical Research (C.C.), Melbourne 3168, Australia

Address all correspondence and requests for reprints to: Dr. Chen Chen, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: chen.chen{at}med.monash edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
GH-releasing peptides (GHRP) are synthetic peptides exerting GH-dependent or GH-independent effects via GH secretagogue receptor on many organs, including the heart. The underlying mechanisms of the cardiotropic properties of GHRP are poorly understood. This study investigates these effects of four GHRP in isolated perfused heart preparations and isolated neonatal and adult ventricular myocytes. The calcium response of cardiocytes to GHRP was visualized using confocal microscopy. All tested GHRP facilitated both ventricular contraction and relaxation in a dose-dependent manner, moderately decreasing coronary flow, but not modifying heart rate. GHRP induced a biphasic increase in intracellular free Ca2+ of the cardiocytes, consisting of a transient phase (phase 1), followed by a plateau phase (phase 2). Phase 1 was abolished by pretreatment with thapsigargin, a Ca2+-adenosine triphosphatase inhibitor of the sarcoplasmic reticulum. The phase 2 response was eliminated by removing extracellular free Ca2+, by verapamil, a voltage-gated Ca2+ channel blocker, or by 24-h pretreatment with phorbol 12-myristate 13-acetate, down-regulating protein kinase C. In isolated (denervated) heart, GHRP have a direct cardiotropic, without chronotropic, effect. GHRP elevate myocardial intracellular free Ca2+ through activating Ca2+ influx via voltage-gated Ca2+ channels and triggering Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores. Protein kinase C mediates the GHRP-induced Ca2+ influx, but not Ca2+ release. These finding support a number of roles for GHRP in the cardiovascular system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
GH-RELEASING peptides (GHRP) are a class of synthetic peptidyl GH secretagogues originally found to stimulate pituitary GH release (1). GH secretagogue receptor (GHS-R), a member of the G protein-coupled receptor family using Gq11 and phosphoplipase C (PLC)-inositol trisphosphate (InsP3) signaling pathways, was first discovered in hypothalamus and pituitary (2). GHS-R has also been identified in several peripheral tissues other than the hypothalamus-pituitary system, particularly in the myocardium, where it probably mediates GH-independent activities (3). Available data suggest that hexarelin (a modified GHRP-6) exerts a GH-independent cardiotropic effect in normal subjects, patients with GH deficiency, and patients with idiopathic or ischemic dilated cardiomyopathy (4). Hexarelin was also reported to protect aged rat heart from postischemic ventricular dysfunction (5). In addition, pretreatment with GHRP-2 has been shown to be protective against the diastolic dysfunction of myocardial stunning in blood-perfused isolated rabbit heart (6). These observations suggest that GHRP may be valuable as potential therapeutic agents for treating heart failure or myocardial stunning. However, little is understood regarding the mechanisms underlying both class effects and individual cardiac effects of GHRP, such as GHRP-1, -2, and -6 and hexarelin. Furthermore, the effects of GHRP on myocardial Ca2+ handling have rarely been studied. This study investigated the direct cardiotropic effects and the underlying Ca2+-mobilizing mechanisms of four GHRP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Constant pressure Langendorff preparation
This study was performed with a total of 40 normal male Sprague Dawley rats, 10–12 wk old, with a mean (±SD) body weight of 242 ± 13 g. The animals were randomly allocated to 5 groups (n = 8/group): GHRP-1 group, GHRP-2 group, GHRP-6 group, hexarelin group, and positive control group (treated with 10-6 mol/liter isoprenaline; catecholamine showing positive inotropic and chronotropic effects on heart). All animals received humane care in compliance with the standards endorsed by the ethics committee of Peking Union Medical College.

The animals were anesthetized with pentobarbital, and the hearts were quickly removed and mounted on a nonrecirculating Langendorff apparatus. Retrograde perfusion was established at a constant pressure of 110 cm H2O with modified Krebs-Henseleit bicarbonate buffer. The perfusion buffer consisted of 118 mmol/liter NaCl, 4.7 mmol/liter KCl, 1.2 mmol/liter CaCl2, 1.2 mmol/liter MgSO4, 25 mmol/liter NaHCO3, 1.2 mmol/liter KH2PO4, and 11.1 mmol/liter glucose. The solution was equilibrated with 95% O2 and 5% CO2 to achieve pH 7.4 at 37 C. Heart temperature was indicated by the temperature of the immediate outflow of the perfused medium from the heart. Heart temperature was 36.7–36.8 C. Apical force displacement (7) and intraventricular balloon were used for monitoring cardiac contraction and relaxation. During apical force displacement, a 5/0 silk ligature was attached to the left ventricular apex and connected to a force transducer (Shuangxing Instrument Co., Tianjing, China). A resting tension of 2.0 g was applied to allow an appropriate recording of both contraction and relaxation. The intraventricular balloon consisted of a handmade emulsion balloon inserted into the left ventricle (LV) via an incision in the left atrium. The balloon was filled with 0.2 ml distilled water and connected to a pressure transducer (Shuangxing Instrument Co., Tianjing, China) via a plastic tube. The transducer output was recorded continuously on a computer-assisted PC-Lab system (Veixinsida Ltd. Technology Co., Beijing, China). After a stabilization time of 10 min, LV end-systolic tension (EST), LV end-diastolic tension (EDT), LV systolic pressure, LV diastolic pressure, and natural heart rate (HR; beats per minute) were recorded. Coronary flow rate (CFR) was estimated by measuring the outflow perfuse medium volume per minute per gram wet weight. In this study constant pressure Langendorff perfusion was used, and as such, the coronary perfusion pressure (CPP) was considered constant.

Measurement of cardiac lactate/pyruvate ratio
Cardiac lactate and pyruvate production were indicated by the levels of lactate and pyruvate in the outflow-perfused medium. Lactate and pyruvate were measured by spectrophotometry using lactate and pyruvate measurement kits (Institute of Nanjing Jiancheng Bioengineering, Nanjing, China). The lactate/pyruvate ratio was used to express the intensity of cardiac anaerobic glycolysis.

Cell culture and intracellular free Ca2+ ([Ca2+]i) measurement
Neonatal cardiomyocytes were obtained from the LV of 1- to 3-d old Sprague Dawley rats, harvested, and digested with collagenase II (1 mg/ml). Cardiomyocytes were purified with differential attachment technique and further identified by sarcomeric {alpha}-actin immunostaining. Primary culture of myocytes was performed using DMEM for 3–4 d. Adult cell studies were performed on enzyme-dissociated single LV cardiocytes of 8- to 10-wk-old Sprague Dawley rats. These cells were not cultured.

The [Ca2+]i response was measured in both cultured myocytes and adult cells loaded with Fluo-3/AM (2 µmol/liter) for 60 min at 37 C. Intracellular fluorescence intensity reflecting [Ca2+]i was determined at room temperature by a confocal microscope (Insight Plus-IQ, Meridian Instruments, Inc., Meridian, CT) at an image rate of 13/sec.

Statistic analysis
All data were expressed as the mean ± SD. Comparison among groups was made using ANOVA. Data obtained in single cell preparations before and after GHRP treatment were analyzed by paired t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Effects of GHRP on coronary flow
GHRP moderately decreased CFR in a dose-dependent manner (Fig. 1Go). The CFR was decreased roughly by 10–15% at maximal dose (10-6 mol/liter). Isoprenaline significantly increased CFR (baseline, 6.5 ± 0.7 ml/min·g; 2 min after 10-6 mol/liter isoprenaline, 8.4 ± 0.5 ml/min·g).



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  		FIG. 1. Dose-response curves showing the effects of GHRP on CFR. CFR decreased (by ~10–15%) at the highest tested GHRP concentration (10-6 mol/liter; n = 8 for each group; mean ± SD).

 
Effects of GHRP on cardiac lactate/pyruvate ratios
The content of lactate in the outflow-perfused medium moderately increased, and pyruvate decreased in all GHRP groups. As such, the cardiac lactate/pyruvate ratios increased (~10–15%) after each GHRP perfusion (Fig. 2Go).



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  		FIG. 2. Effects of GHRP on cardiac lactate/pyruvate (L/P) ratios. Each of four GHRP significantly increased L/P ratios (n = 8 for each group; mean ± SD).

 
Effects of GHRP on heart rate
No significant effect on HR in isolated (denervated) heart in response to GHRP was observed when recorded by apical displacement (Table 1Go and Fig. 3AGo). HR was accelerated by approximately 25% (n = 4) when recorded by intraventricular balloon (Fig. 3BGo). Isoprenaline significantly increased HR (Table 1Go).


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  		TABLE 1. Effects of GHRPs (10-6 M) and isoprenaline (10-6 M) on heart rate (beats/min)

 


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  		FIG. 3. Original cardiac beat traces showing the positive cardiotropic effects of GHRP. Note that GHRP facilitated both ventricular contraction and relaxation. Fast-run traces at the right of each subpanel show trace configurations of cardiac beating recorded by two different methods. HR was not modulated when recorded by apical displacement (A). HR was slightly accelerated by GHRP when recorded by intraventricular balloon (B). The time scales in four subpanels of A or B are the same as indicated in the figure.

 
Cardiotropic effect of GHRP
GHRP facilitated both ventricular contraction and relaxation when recorded by apical displacement (Fig. 3AGo) and intraventricular balloon (Fig. 3BGo). EST increased and EDT decreased after each GHRP perfusion. Table 2Go shows the changes in EST and EDT for each group 5 min after GHRP (10-8 mol/liter, maximal response) or isoprenaline (10-6 mol/liter) injection. The dose analysis was performed with a range of GHRP between 5 x 10-11 and 10-6 mol/liter (Fig. 4Go). Isoprenaline at 10-6 mol/liter exerted a higher cardiotropic effect than any GHRP (Table 2Go). At baseline, cardiac beating was stable, with no observable run-down phenomenon within 2 h.


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  		TABLE 2. Effects of GHRPs (10-8 mol/liter) and isoprenaline (10-6 mol/liter) on cardiac contraction and relaxation (n = 8 for each group)

 


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  		FIG. 4. Cumulative dose-response curves of ESTs (upper panel) and EDTs (lower panel) in response to GHRP (n = 8 for each group; mean ± SD).

 
Ca2+-mobilizing effects of GHRP on cultured neonatal cardiomyocytes
GHRP induced a biphasic [Ca2+]i response in approximately 90% of cultured neonatal cells, consisting of a spike-like transient phase (phase 1), followed by a plateau phase (phase 2; Fig. 5Go). The Ca2+ transient appeared immediately (2–3 sec) after injecting GHRP into the cell chamber and reached a peak 140–150 sec after the injection. At baseline, the background fluorescent intensity of cardiomyocytes were relatively low, and no spontaneous [Ca2+]i oscillation was observed.



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  		FIG. 5. Ca2+-mobilizing effects of GHRP on cultured neonatal rat cardiomyocytes. The three left columns are representative confocal fluo-3 fluorescent images at baseline, 142 sec (peak response), and 462 sec after GHRP (10-8 mol/liter) exposure, respectively. The right column shows the respective [Ca2+]i dynamic traces of the single cells marked by the white circles and the number 1. The blue straight lines mark the injection of GHRP into the sample chamber. The white lines under each trace indicate the period (in seconds) of GHRP exposure.

 
Verapamil (10-6 mol/liter) did not affect the phase 1, but totally abolished the phase 2, response in Hanks ’ solution (Fig. 6Go). In addition, removing extracellular Ca2+ ([Ca2+]o; using Ca2+-free Dulbecco’s Hank’s solution and 5 mmol/liter EGTA) decreased the phase 1 elicited by any GHRP (Fig. 6).



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  		FIG. 6. Representative confocal images showing the effects of verapamil, [Ca2+]o, thapsigargin, and PMA on [Ca2+]i responses induced by GHRP. Note that pretreatment with verapamil did not affect the phase 1, but abolished the phase 2, response. Removing [Ca2+]o decreased the phase 1 and abolished the phase 2 response. Pretreatment with thapsigargin abolished the phase 1, but did not depress the phase 2, response. Pretreatment with PMA did not affect the phase 1, but abolished the phase 2, response induced by GHRP.

 
Pretreatment with thapsigargin (100 nmo/liter) for 45 min depleted intracellular Ca2+ stores and abolished the phase 1, but did not affect the phase 2, response to GHRP. Pretreatment with phorbol 12-myristate 13-acetate (PMA; 10-6 mol/liter) for 24 h, depleting protein kinase C (PKC), did not affect the phase 1, but abolished the phase 2, response to GHRP (Fig. 6Go).

Acute injection of thapsigargin (final concentration, 5 x 10-5 mol/liter) induced a significant Ca2+ transient in neonatal cardiocytes. After the thapsigargin-induced Ca2+ transient, GHRP induced an additional significant Ca2+ transient (Fig. 7Go).



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  		FIG. 7. Representative confocal images showing the Ca2+ transients induced by acute injection of thapsigargin and hexarelin in cultured neonatal cardiocyte. A, Baseline; B, peak response to thapsigargin (10-6 mol/liter); C, 100 sec after thapsigargin injection; D, peak response to hexarelin (10-8 mol/liter); E, 500 sec after hexarelin injection; F, [Ca2+]i dynamic trace corresponding to the cell at which the white arrow is pointed. The blue lines marked by 1 and 2 are markers of thapsigargin and hexarelin injection, respectively.

 
Acute injection of caffeine (final concentration, 10-3 mol/liter) to the cell culture medium did not induce a significant Ca2+ transient in cultured neonatal cardiocytes. Further, a Ca2+ transient was not observed after extreme doses of caffeine (Fig. 8Go). However, treatment with hexarelin (10-8 mol/liter) induced a biphasic Ca2+ transient with a high magnitude in cultured neonatal cardiocytes after caffeine injection (Fig. 8Go).



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  		FIG. 8. Representative confocal images showing the caffeine- and hexarelin-induced Ca2+ transients in neonatal cardiomyocytes. A, Baseline; B, 40 sec after the caffeine (10-3 mol/liter) injection. Note that caffeine could not induce significant Ca2+ transient; C, 40 sec after an extra dose of caffeine, there was still no Ca2+ response; D, peak response to hexarelin (10-8 mol/liter); E, 300 sec after hexarelin injection; F, [Ca2+]i dynamic trace corresponding to the cell at which the white arrow is pointed. The blue lines marked by 1, 2, and 3 are markers of the two caffeine and single hexarelin injections, respectively.

 
Ca2+-mobilizing effects of GHRP on adult cardiomyocytes
Hexarelin induced a biphasic Ca2+ transient in adult enzyme-dissociated cardiomyocytes (Fig. 9Go). In addition, after the significant Ca2+ transient in adult cardiocytes induced by caffeine (10-3 mol/liter), hexarelin was able to induce a significant Ca2+ transient (Fig. 10Go).



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  		FIG. 9. Representative confocal images showing the Ca2+-mobilizing effect of hexarelin on adult, enzyme-dissociated rat cardiomyocyte. A, Baseline; B, peak response to hexarelin (10-8 mol/liter); C, 300 sec after hexarelin injection; D, [Ca2+]i dynamic trace. The blue line is the marker of hexarelin injection. The other three GHRP induced similar Ca2+ responses, but data were not shown.

 


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  		FIG. 10. Representative confocal images showing the caffeine- and hexarelin-induced Ca2+ transients in adult, enzyme-dissociated rat cardiomyocyte. A, Baseline; B, peak response to caffeine (10-3 mol/liter); C, 60 sec after caffeine injection; D, peak response to hexarelin (10-8 mol/liter); E, 200 sec after hexarelin injection; F, [Ca2+]i dynamic trace corresponding to the cell at which the white arrow is pointed. The blue lines marked by 1 and 2 are markers of caffeine and hexarelin injections, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this study we present data showing the potential importance of GHRP in the rat heart, particularly with respect to inotropic and calcium-mobilizing effects. GHRP (namely GHRP-1, -2, and -6 and hexarelin) have direct cardiotropic effects, including facilitation of both ventricular contraction and relaxation. In adult cardiocytes these agents induced biphasic Ca2+ transient. In cultured neonatal cardiocytes we demonstrate GHRP elevate myocardial [Ca2+]i through activating Ca2+ influx via voltage-gated Ca2+ channel and triggering Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores. In addition we show that PKC mediates GHRP-induced Ca2+ influx, but not Ca2+ release, GHRP moderately decrease CFR, without inhibiting their cardiotropic effect and do not modulate HR in isolated (denervated) heart.

Considering that GHRP have no significant chronotropic effect or obvious toxicity, it is possible that these agents may be developed further to treat myocardial dysfunction such as heart failure. Future studies performed to define GHRP influence on coronary perfusion and other side-effects would be required to determine the extent of any GHRP therapeutic effect.

Effect of GHRP on coronary resistance
In the present study we demonstrate that GHRP elicit a modest decrease in CFR at constant perfusion pressure. Bodart et al. (8) reported that hexarelin significantly increased CPP in isolated, Langendorff-perfused rat heart under constant perfusion flow, proposing that this agent exerted its coronary constricting effect at least in part by L-type Ca2+ channels and PKC. The notable increase in CPP was achieved only at very high concentrations of hexarelin (5 x 10-5 to 5 x 10-4 mol/liter) in Bodart’s study, concentrations unlikely to be used clinically. In both the present and the previous study, a GHRP concentration of 10-6 mol/liter results in a moderate (~10%-15%) change in both CFR and CPP, respectively. Interestingly, as coronary flow velocity may increase when the coronary artery is restricted, an increase in coronary resistance may not necessarily evoke a proportional decrease in coronary flow rate. Generally, coronary flow will only decrease significantly when the coronary artery is constricted to less than one quarter of the control value.

The data presented in this study suggest that the coronary constricting effect of GHRP does not inhibit their cardiotropic effect, a phenomena that may lead to an imbalance in oxygen supply and consumption in the myocardium. To investigate the extent of this effect we measured myocardial lactate/pyruvate ratios. The baseline lactate/pyruvate ratio (before GHRP infusion) was comparable with that in normal tissue reported by Frykholm et al. (9), indicating that there was no cardiac hypoxia in the isolated Langendorff-perfused heart before GHRP infusion. Our results demonstrate that GHRP at a concentration 10-6 mol/liter increase the lactate/pyruvate ratio by approximately 10–15%. An increase in the lactate/pyruvate ratio reflects a degree of anaerobic glycolysis occurring in the myocardium during GHRP treatment. However, as this level is significantly less than that measured during tissue ischemia (9), we speculate that GHRP infusion at this concentration induces moderate hypoxia or ischemia. The mechanisms underlying this effect may include the cardiotropic effect of GHRP, whereby an increase in oxygen consumption occurs under the influence of GHRP. Minor hypoxia due to coronary constriction and/or bad perfusion in some areas of the heart also cannot be excluded.

Effect of GHRP on heart rate
In the present study we show that GHRP have no significant effect on the HR of isolated (denervated) heart at doses producing maximal cardiotropic response (10-8 mol/liter) and above (10-6 mol/liter) when recorded by apical displacement. However, when recorded by the intraventricular balloon method, GHRP accelerated the HR by approximately 25%. This phenomenon is not likely to be physiologically significant and more probably is due to improper stretching of the myocardium by the handmade balloon or incision of the left atrium when inserting the balloon into the LV, affecting the automaticity of myocardium in the intraventricular balloon method. It has been previously reported (8) that GHRP (hexarelin) does not modify HR in isolated rat heart, and studies by Bisi et al. (10) have shown that hexarelin increased LVEF without significant changes in mean blood pressure or HR in human subjects. Although further investigations are needed to define the extent of the chronotropic effect of GHRP, this study supports existing evidence that GHRP (in particular, hexarelin) exerts no significant influence on HR. The absence of any chronotropic effect of GHRP decreases associated physiological risks, such as increased cardiac oxygen consumption with accelerated HR or arrhythmias related to rapid or slow HR, and provides support for a therapeutic basis for these agents.

Mechanisms of the cardiotropic effect of GHRP
The influence of GHRP on the heart has been shown to be mediated by both GHS-R and CD36 (11). Recently, GHRP-binding sites were identified in the heart (3), and CD36, a multifunctional B-type scavenger receptor, has been reported to mediate the cardiac action of hexarelin (12). We confirm reports by others (4, 5, 6) suggesting that the cardiotropic influence of GHRP is direct (possibly GHS-R and CD36 mediated) rather than GH dependent, as GH does not show acute cardiotropic action (10) and does not affect cardiac systolic function even administrated chronically (13). However, because GH exerts long-term cardiotrophic effect and regulates cardiac metabolic homeostasis (14), and GHRP does induce GH release, the GH-dependent component of the GHRP (possibly ghrelin also) action on the heart cannot be excluded under in vivo conditions, which has been reported to play an important role in the regulation of metabolic balance and in the prevention of cardiac cachexia through GH-dependent or -independent mechanisms (15).

Interestingly, Bedendi et al. (16) reported that the effects of hexarelin on cardiomyocytes are mediated predominantly by endothelium-released prostaglandin I2(PGI2). Cardiac cell studies in the present study, however, show that GHRP exert a significant calcium-mobilizing effect in the absence of endothelium, suggesting the existence of PGI2-independent pathways in the myocardium. GH-independent effects of GHRP are mediated by GHS-R with downstream signaling molecules, including Gq protein, PLC, InsP3, and PKC. PGI2 may also be one of the downstream signaling molecules. Thus, it appears that the cardiac actions of GHRP might include both PGI2-dependent and PGI2-independent mechanisms.

[Ca2+]i elevation in cardiomyocytes is a key factor signaling the initiation of cardiac contraction. Although GHRP has been reported to induce [Ca2+]i elevation in pituitary cells (17), little is known regarding the action of GHRP on the calcium handling of cardiomyocytes. We show that GHRP induce biphasic [Ca2+]i elevation through activating Ca2+ influx and triggering Ca2+ release from the intracellular Ca2+ store(s) in cultured neonatal and enzyme-dissociated adult cardiomyocytes. This Ca2+-mobilizing effect of GHRP may contribute to their cardiotropic effects.

Ca2+-mobilizing effects of GHRP in neonatal and adult cardiomyocytes
Neonatal cardiocytes use very different mechanisms to regulate calcium homeostasis compared with adult cardiocytes. Previously, neonatal myocytes were observed to rely on both extracellular and nuclear calcium for contractile function, whereas freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function (18). However, culture of adult cardiomyocytes leads them to develop additional mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes (18). In the present study we show that GHRP stimulate both Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from intracellular Ca2+ store in neonatal cardiocytes. Although the subcellular localization of the intracellular store was not identified in the present study, we demonstrate that after Ca2+ depletion with chronic thapsigargin (a Ca2+-adenosine triphosphatase inhibitor of the sarcoplasmic reticulum) treatment in neonatal cardiocytes, the phase 1 response (Ca2+ release) was abolished, but the phase 2 response (Ca2+ influx) was not affected by GHRP. Acute use of thapsigargin induced a significant [Ca2+]i elevation. Based on previous evidence (18) and the present data, we hypothesize that the nuclear Ca2+ store in neonatal cardiocytes is also thapsigargin sensitive.

Intracellular Ca2+ stores include both InsP3-sensitive and caffeine-sensitive Ca2+ reserves (19). It is unclear which of the two stores is predominantly responsible for the calcium-mobilizing action of GHRP in the neonatal cardiocyte. In this study we present data to show that caffeine does not induce significant Ca2+ mobilization in cultured neonatal cardiocytes, whereas hexarelin induces a significant Ca2+ response after caffeine injection, suggesting that the caffeine-sensitive Ca2+ store is underdeveloped and plays a minor role in GHRP-induced calcium mobilization in neonatal cardiocytes. The thapsigargin-sensitive Ca2+ stores appear to be the primary source of GHRP-induced Ca2+ release in neonatal cardiocytes. It still remains unclear whether this thapsigargin-sensitive Ca2+ store is also InsP3 sensitive in the neonatal cardiocyte.

In this study we also present evidence that GHRP induces a biphasic Ca2+ transient in the adult cardiocyte. GHRP may trigger Ca2+ release from the sarcoplasmic reticulum and possibly stimulate Ca2+ influx through L-type Ca2+ channels (18). GHRP-6 has been reported to induce a biphasic [Ca2+]i response in rat pituitary somatotropes (20). Here, we demonstrated for the first time that GHRP induce a biphasic elevation of [Ca2+]i in both neonatal and adult cardiomyocytes.

Investigation into the mechanisms of GHRP-induced Ca2+ mobilization in neonatal cardiocytes suggests that phase 1 of the biphasic elevation is due mainly to Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores. We show that pretreatment with thapsigargin abolishes the phase 1 response. Furthermore, phase 2 is probably elicited by Ca2+ influx via voltage-gated Ca2+ channels, as verapamil (a voltage-gated Ca2+ channel blocker) or Ca2+-free medium eliminates this effect. The phenomenon that removing extracellular Ca2+ slightly decreased the phase 1 response may be due to the attenuation of the Ca2+-induced Ca2+ release (21) or a reduction in Ca2+ stores due to disrupted Ca2+ replenishment.

GHS-R, which mediates the actions of both endogenous GHS (ghrelin) and synthetic peptidyl (GHRP) or nonpeptidyl GHS, has been found to include two subtypes: GHS-R1a, the functional receptor, and GHS-R1b, an unspliced, nonfunctional GHS-R (2, 3, 22). Both subtypes are widespread in almost all tissues. It is likely that the cardiovascular effects of GHRP are mediated by GHS-R1a. The function of GHS-R1b is relatively understudied, and its role in mediating the cardiac effect of GHRP requires further investigation. Varieties of intracellular signaling are thought to be involved in the GHRP mechanism, which include changes in [Ca2+]i, cAMP, PKA, PKC, and PLC (23). To date signaling engaged by GHRP has been explored predominantly in pituitary cells and understudied in cardiomyocytes. The present study suggests that in cardiomyocytes, the signaling molecules that relate to Ca2+ influx and Ca2+ release from thapsigargin-sensitive intracellular stores may be involved in GHRP-induced [Ca2+]i elevation. We further demonstrate that PKC mediates GHRP-induced Ca2+ influx, but not Ca2+ release.

Relationship between acute and chronic administration, and the cardioprotective effect of GHRP
Cardiac dysfunction in myocardial stunning and heart failure consists of impaired systolic and diastolic function and deficient [Ca2+]i handling. Increasingly, data suggest that some GHRP (in particular, GHRP-2 and hexarelin) show cardioprotective effects in ischemic myocardial stunning and cardiomyopathy (4, 5, 6). However, most studies investigating the cardioprotective effects of GHRP have been performed using chronic administration (long or short term) in both animal and human subjects (4, 5, 6, 24, 25, 26, 27, 28, 29). When treating cardiac pumping dysfunction, the cardiotropic effect of GHRP could be considered cardioprotective. The effects of acute administration of GHRP and whether these agents exert a similar action as when chronically administered have not been studied and are basically unknown. We show in the present study that GHRP facilitate both ventricular contraction and relaxation in normal heart, and we speculate that as such, GHRP may be beneficiary in treating both systolic and diastolic cardiac dysfunction. However, because the focus of the present study was to investigate the cardiotropic and calcium-mobilizing effects of GHRP on normal heart, we are not able at this stage to relate these effects qualitatively with cardioprotection.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
This paper describes the direct positive inotropic effect, without an obvious chronotropic effect, that GHRP exertd on rat heart. GHRP elevate myocardial [Ca2+]i via stimulating Ca2+ influx through the voltage-gated Ca2+ channel and triggering Ca2+ release from thapsigargin-sensitive intracellular Ca2+ store(s). This Ca2+-mobilizing effect of GHRP may underlie their documented cardiotropic effects. GHRP moderately decrease coronary flow. However, this effect does not prohibit their cardiotropic action. These findings are important, as GHRP mechanisms within the cardiovascular system are poorly understood. Future studies investigating these mechanisms and examining possible physiological side-effects may add new information to the potential clinical perspectives for GHRP as cardiotropic agents.


   Acknowledgments
 
We thank Dr. Guang-Hong Bao, Dr. Yong-Fang Zheng, Wei Hao, and Dejie Yu for their assistance. GHRP-2 was provided by Dr. C. Y. Bowers (Tulane Medical Center, New Orleans, LA), and hexarelin by Dr. R. Deghenghi (Europeptide, Argenteuil, France). GHPR-1 was kindly supplied by Kaken Pharmaceutical Co. (Tokyo, Japan). GHRP-6 was purchased from Sigma-Aldrich Pty. Ltd. (New Castle, Australia).


   Footnotes
 
This work was supported by Distinguished Young Investigator Awards (30028007 to C.C.; 30125016 to J.-M.C.) and grants (39970300 to J.-M.C.; 30270507 to R.-K.X.) from the Natural Sciences Foundation of China.

X.-B.X. and J.-M.C. contributed equally to this work.

C.C. is a senior research fellow supported by the Australian National Health and Medical Research Council.

Abbreviations: [Ca2+]i, Intracellular free Ca2+; [Ca2+]o, extracellular free Ca2+; CFR, coronary flow rate; CPP, coronary perfusion pressure; EDT, end-diastolic tension; EST, end-systolic tension; GHRP, GH-releasing peptides; GHS-R, GH secretagogue receptor; HR, heart rate; InsP3, inositol trisphosphate; LV, left ventricle; PGI2, prostaglandin I2;PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate.

Received January 7, 2003.

Accepted for publication August 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Bowers CY, Momany F, Reynolds GA, Chang D, Hong A, Chang K 1980 Structure-activity relationships of a synthetic pentapeptide that specifically releases growth hormone in vitro. Endocrinology 106:663–667[Abstract/Free Full Text]
  2. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH 1996 A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974–977[Abstract]
  3. Papotti M, Ghè C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, Muccioli G 2000 Growth hormone secretagogues binding sites in peripheral human tissues. J Clin Endocrinol Metab 85:3803–3807[Abstract/Free Full Text]
  4. Broglio F, Benso A, Valetto MR, Gottero C, Quaranta L, Podio V, Arvat E, Bobbio M, Bisi G, Ghigo E 2001 Growth hormone-independent cardiotropic activities of growth hormone-releasing peptides in normal subjects, in patients with growth hormone deficiency, and in patients with idiopathic or ischemic dilated cardiomyopathy. Endocrine 14:105–108[CrossRef][Medline]
  5. Rossoni G, De Gennaro Colonna V, Bernareggi M, Polvani GL, Muller EE, Berti F 1998 Protectant activity of hexarelin or growth hormone against postischemic ventricular dysfunction in hearts from aged rats. J Cardiovasc Pharmacol 32:260–265[CrossRef][Medline]
  6. Weekers F, Van Herck E, Isgaard J, Van den Berghe G 2000 Pretreatment with growth hormone-releasing peptide-2 directly protects against the diastolic dysfunction of myocardial stunning in an isolated, blood-perfused rabbit heart model. Endocrinology 141:3993–3999[Abstract/Free Full Text]
  7. Dogan R, Farsak B, Isbir S, Sarigul A, Tuncer M, Kilinc K 2001 Prospective effect of lisinopril against ischemia-reperfusion injury in isolated guipea pig hearts. J Cardiovasc Surg 42:43–48[Medline]
  8. Bodart V, Bouchard JF, McNicoll N, Escher E, Carrière P, Ghigo E, Sejlitz T, Sirois MG, Lamontagne D, Ong H 1999 Identification and characterization of a new growth hormone-releasing peptide receptor in the heart. Circ Res 85:796–802[Abstract/Free Full Text]
  9. Frykholm P, Hillered L, Långström B, Persson L, Valtysson J, Watanabe Y, Enblad P 2001 Increase of interstitial glycerol reflects the degree of ischaemic brain damage: a PET and microdialysis study in a middle cerebral artery occlusion-reperfusion primate model. J Neurol Neurosurg Psychiatry 71:455–461[Abstract/Free Full Text]
  10. Bisi G, Podio V, Valetto MR, Broglio F, Bertuccio G, Del Rio G, Arvat E, Boghen MF, Deghenghi R, Muccioli G, Ong H, Ghigo E 1999 Acute cardiovascular and hormonal effects of GH and hexarelin, a synthetic GH-releasing peptide, in humans. J Endocrinol Invest 22:266–272[Medline]
  11. Torsello A, Bresciani E, Rossoni G, Avallone R, Tulipano G, Cocchi D, Bulgarelli I, Deghenghi R, Berti F, Locatelli V 2003 Ghrelin plays a minor role in the physiological control of cardiac function in the rat. Endocrinology 144:1787–1792[Abstract/Free Full Text]
  12. Bodart V, Febbraio M, Demers A, McNicoll N, Pohankova P, Perreault A, Sejlitz T, Escher E, Silverstein RL, Lamontagne D, Ong H 2002 CD36 mediates the cardiovascular action of growth hormone-releasing peptides in the heart. Circ Res 90:844–849[Abstract/Free Full Text]
  13. Cittadini A, Isgaard J, Monti MG, Casaburi C, Di Gianni A, Serpico R, Iaccarino G, Sacca L 2003 Growth hormone prolongs survival in experimental postinfarction heart failure. J Am Coll Cardiol 41:2154–2163[Abstract/Free Full Text]
  14. Amato G, Carella C, Fazio S, La Montagna G, Cittadini A, Sabatini D, Marciano-Mone C, Sacca L, Bellastella A 1993 Body composition, bone metabolism, heart structure and function in growth hormone deficient adults before and after growth hormone replacement therapy at low doses. J Clin Endocrinol Metab 77:1671–1676[Abstract]
  15. Nagaya N, Uematsu M, Kojima M, Ikeda Y, Yoshihara F, Shimizu W, Hosoda H, Hirota Y, Ishida H, Mori H, Kangawa K 2001 Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation 104:1430–1435[Abstract/Free Full Text]
  16. Bedendi I, Gallo MP, Malan D, Levi RC, Alloatti G 2001 Role of endothelial cells in modulation of contractility induced by hexarelin in rat ventricle. Life Sci 69:2189–2201[CrossRef][Medline]
  17. Deghenghi R, Cananzi MM, Torsello A, Battisti C, Muller EE, Locatelli V 1994 GH-releasing activity of hexarelin, a new growth hormone releasing peptide, in infant and adult rats. Life Sci 54:1321–1328[CrossRef][Medline]
  18. Poindexter BJ, Smith JR, Buja LM, Bick RJ 2001 Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal and adult cardiomyocytes. Cell Calcium 30:373–382[CrossRef][Medline]
  19. Pozzan T, Rizzuto R, Volpe P, Meldolesi J 1994 Molecular and cellular physiology of intracellular calcium stores. Physiol Rev 74:595–636[Free Full Text]
  20. Ghigo E, Arvat E, Muccioli G, Camanni F 1997 Growth hormone releasing peptides. Eur J Endocrinol 136:445–460[Abstract/Free Full Text]
  21. Cheng H, Wang SQ 2002 Calcium signaling between sarcolemmal calcium channels and ryanodine receptors in heart cells. Front Biosci 7:D1867–D1878
  22. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M 2002 The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 87:2988–2991[Abstract/Free Full Text]
  23. Chen C 2000 Growth hormone secretagogue actions on the pituitary gland: multiple receptors for multiple ligands? Clin Exp Pharmacol Physiol 27:323–329[CrossRef][Medline]
  24. Muccioli G, Broglio F, Valetto MR, Ghe C, Catapano F, Graziani A, Papotti M, Bisi G, Deghenghi R, Ghigo E 2000 Growth hormone-releasing peptides and the cardiovascular system. Ann Endocrinol (Paris) 61:27–31[Medline]
  25. Berti F, Muller E, De Gennaro Colonna V, Rossoni G 1998 Hexarelin exhibits protective activity against cardiac ischaemia in hearts from growth hormone-deficient rats. Growth Horm IGF Res 8(Suppl B):149–152.
  26. Rossoni G, Locatelli V, Gennaro Colonna Vd, Muller EE, Berti F 2000 Hexarelin, a growth hormone secretagogue, protects the isolated rat heart from ventricular dysfunction produced by exposure to calcium-free medium. Pharmacol Res 42:129–136[CrossRef][Medline]
  27. Torsello A, Rossoni G, Locatelli V, De Gennaro Colonna V, Bernareggi M, Francolini M, Muller EE, Berti F 2001 Hexarelin, but not growth hormone, protects heart from damage induced in vitro by calcium deprivation replenishment. Endocrine 14:109–112[CrossRef][Medline]
  28. Locatelli V, Rossoni G, Schweiger F, Torsello A, De Gennaro Colonna V, Bernareggi M, Deghenghi R, Muller EE, Berti F 1999 Growth hormone-independent cardioprotective effects of hexarelin in the rat. Endocrinology 140:4024–4031[Abstract/Free Full Text]
  29. Tivesten A, Bollano E, Caidahl K, Kujacic V, Sun XY, Hedner T, Hjalmarson A, Bengtsson BA, Isgaard J 2000 The growth hormone secretagogue hexarelin improves cardiac function in rats after experimental myocardial infarction. Endocrinology 141:60–66[Abstract/Free Full Text]



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